Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells ofGlycinemax

Abstract

Polyribosome formation was stimulated by cytokinin treatment of cultured cells of Glycine max cv. Funk Delicious. When suspension cultures were given 0-5 pM zeatin after 24 h in culture in medium lacking a cytokinin, a nearly 2-fold increase in the polyribosome/monoribosome ratio occurred over the subsequent 3 h. The effect of actinomycin D and of 5-fluorouridine on RNA synthesis and on the polyribosome/monoribosome ratios of these cells was examined. Actinomycin D at 5 and 20 pg/ml_1inhibited total RNA synthesis by 39 and 60%, respectively, as measured by [3H]uridine incorporation into acid-precipitable material. The degree of inhibition of precursor incorporation into polyribosomal RNA was similar. At 0-1 mM, 5 fluorouridine inhibited [3H]uridine incorporation by 76%, and [3H]guanosine incorporation by 66% into polyribosomal RNA after 3 h of treatment. Fractionation of the polyribosomal RNA by oligo(dT)-cellulose chromatography demonstrated that low concentrations of both actinomycin D (5 pg ml-1) and 5-fluorouridine (0-1 mM) inhibited the synthesis of ribosomal RNA to a greater extent than the poly(A)-containing fraction of the messenger RNA. Synthesis of the poly (A) -containing RNA was inhibited by 24% with 5 pg ml-1 actinomycin D and by 30% with 0-1 mM 5-fluorouridine. At the above concentrations, these two inhibitors reduced the polyribosome/monoribosome ratio of the cytokinin-deprived cells over a 3 h period, but they did not prevent cytokinin-induced polyribosome formation. These results provide further evidence that cytokinin regulates polyribosome levels through an effect on protein synthesis at the translational level